# C6-Alkoxy substituted penicillins are potent non-covalently binding inhibitors of the SARS-CoV-2 main protease

## TL;DR

Researchers found that modified penicillin drugs can inhibit a key virus enzyme without forming traditional enzyme-drug complexes, suggesting new ways to treat diseases like COVID-19.

## Contribution

The study introduces C6-alkoxy substituted penicillins as non-covalent inhibitors of SARS-CoV-2 main protease, offering a novel mechanism of action.

## Key findings

- C6-alkoxy substituted penicillins inhibit Mpro via non-covalent interactions at the active site.
- (R)-sulfoxides of these derivatives show covalent reaction with Mpro, unlike (S)-sulfoxides.
- Modifying penicillin with a C6-alkoxy group allows inhibition of Mpro without forming acyl–enzyme complexes.

## Abstract

Inhibition of the SARS-CoV-2 main protease (Mpro) by small-molecules is a validated strategy for COVID-19 treatment. There is a need for improved Mpro inhibitors, including because Mpro mutations can confer resistance to clinically used Mpro inhibitors. Previous work has revealed the potential of penicillin derivatives as covalently reacting Mpro inhibitors. Here we report studies on Mpro inhibition by C6-alkoxy substituted penicillin derivatives. The combined mass spectrometric and computational evidence imply most of the tested penicillin C6-alkoxy derivatives bind via non-covalent interactions at the Mpro active site, resulting in potent substrate-competitive inhibition. Some penicillin C6-alkoxy derivatives ((R)-, but not (S)-sulfoxides) manifest covalent reaction to different extents. Penicillins and related drugs are widely used antibiotics, acting via covalent reaction of their β-lactam with a nucleophilic serine in their transpeptidase targets to give an acyl–enzyme complex. The results imply penicillin derivatives can be developed to inhibit enzymes via mechanisms other than formation of stable acyl–enzyme complexes.

Introduction of a C6-alkoxy group on the penicillin scaffold can alter the mechanism of SARS-CoV-2 main protease (Mpro) inhibition.